Department of Anatomy, Shandong University School of Medicine, Jinan 250012, China.
Department of Cardiosurgery, Shandong University Qilu Hospital, Jinan 250012, China.
Brain Res Bull. 2014 Jan;100:93-106. doi: 10.1016/j.brainresbull.2013.11.007. Epub 2013 Dec 5.
The vesicular glutamate transporter 3 (VGLUT3) and the vesicular monoamine transporter 2 (VMAT2) are expressed in dorsal root ganglion (DRG) neurons and play an important role in packing the neurotransmitter into synaptic vesicles. Brain-derived neurotrophic factor (BDNF) is one of the most profound known regulators of survival in the developing peripheral nervous system (PNS). Whether BDNF regulates the expression of VGLUT3 and VMAT2 in DRG neurons is still unclear. In the present study, primary cultured rat DRG neurons were used to evaluate the effects of BDNF on VGLUT3 and VMAT2 expression. The signaling pathways of the extracellular signal-regulated protein kinase 1/2 (ERK1/2), the phosphatidylinositol 3-kinase (PI3K)/Akt, and the phospholipase C-gamma (PLC-γ) involved in these effects were also determined. DRG neurons at 48h post-culture were incubated with BDNF and/or ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and PLC-γ inhibitor U73122 for an additional 24h. After that, the neurite growth and growth-associated protein 43 (GAP-43) expressions after different doses of BDNF treatment were determined by immunofluorescent labeling. The expression of mRNA and protein of VGLUT3 and VMAT2 in different experimental conditions was assessed by real-time PCR, immunoblotting, and immunofluorescent labeling, respectively. The results showed that BDNF exposure promoted neurite growth and GAP-43 expression in DRG neurons in a dose-dependent manner. BDNF induced VGLUT3 upregulation through activation of PLC-γ signaling pathway. Although BDNF administration did not elevate the levels of VMAT2, the block of the PI3K/Akt or PLC-γ signaling pathways could inhibit VMAT2 expression in DRG neurons in the presence of BDNF. The knockdown of VGLUT3 or VMAT2 gene by siRNA did not affect the BDNF's effects on GAP-43 upregulation and neurite growth. The upregulation of VGLUT3 induced by BDNF might be that BDNF improved neuronal outgrowth status by promoting GAP-43 expression to stimulate neurite elongation. The contribution of distinct VGLUT3 and VMAT2 transporter expression induced by BDNF might be one of the mechanisms that BDNF regulates neuropathic pain. These data imply that BDNF signaling system might be a potential target on modifying distinct transporter-mediated biological effects of primary sensory neurons.
囊泡谷氨酸转运体 3（VGLUT3）和囊泡单胺转运体 2（VMAT2）在背根神经节（DRG）神经元中表达，并在将神经递质包装到突触小泡中发挥重要作用。脑源性神经营养因子（BDNF）是已知的对周围神经系统（PNS）发育中存活具有深远影响的因子之一。BDNF 是否调节 DRG 神经元中 VGLUT3 和 VMAT2 的表达尚不清楚。在本研究中，使用原代培养的大鼠 DRG 神经元来评估 BDNF 对 VGLUT3 和 VMAT2 表达的影响。还确定了涉及这些作用的细胞外信号调节蛋白激酶 1/2（ERK1/2）、磷脂酰肌醇 3-激酶（PI3K）/Akt 和磷酯酶 C-γ（PLC-γ）信号通路。在培养后 48 小时，将 DRG 神经元与 BDNF 和/或 ERK1/2 抑制剂 PD98059、PI3K 抑制剂 LY294002 和 PLC-γ 抑制剂 U73122 孵育 24 小时。之后，通过免疫荧光标记确定不同剂量 BDNF 处理后神经突生长和生长相关蛋白 43（GAP-43）表达情况。通过实时 PCR、免疫印迹和免疫荧光标记分别评估不同实验条件下 VGLUT3 和 VMAT2 的 mRNA 和蛋白表达。结果表明，BDNF 暴露以剂量依赖性方式促进 DRG 神经元的神经突生长和 GAP-43 表达。BDNF 通过激活 PLC-γ 信号通路诱导 VGLUT3 上调。尽管 BDNF 给药不会升高 VMAT2 的水平，但在 BDNF 存在下，PI3K/Akt 或 PLC-γ 信号通路的阻断可抑制 DRG 神经元中 VMAT2 的表达。siRNA 敲低 VGLUT3 或 VMAT2 基因不会影响 BDNF 对 GAP-43 上调和神经突生长的作用。BDNF 诱导的 VGLUT3 上调可能是因为 BDNF 通过促进 GAP-43 表达来改善神经元生长状态，从而刺激神经突伸长。BDNF 诱导的不同 VGLUT3 和 VMAT2 转运体表达的贡献可能是 BDNF 调节神经病理性疼痛的机制之一。这些数据表明，BDNF 信号系统可能是改变初级感觉神经元中不同转运体介导的生物学效应的潜在靶点。
